Effect of infant viral respiratory disease on childhood asthma in a non‐industrialized setting

Abstract Background There are limited data from non‐industrialized settings on the effects of early life viral respiratory disease on childhood respiratory illness. We followed a birth cohort in tropical Ecuador to understand how early viral respiratory disease, in the context of exposures affecting airway inflammation including ascariasis, affect wheezing illness, asthma, and rhinoconjunctivitis in later childhood. Methods A surveillance cohort nested within a birth cohort was monitored for respiratory infections during the first 2 years in rural Ecuador and followed for 8 years for the development of wheeze and rhinoconjunctivitis. Nasal swabs were examined for viruses by polymerase chain reaction and respiratory symptom data on recent wheeze and rhinoconjunctivitis were collected by periodic questionnaires at 3, 5, and 8 years. Stools from pregnant mothers and periodically from children aged 2 years were examined microscopically for soil‐transmitted helminths. Atopy was measured by allergen skin prick testing at 2 years. Spirometry, fractional exhaled nitric oxide measurement, and nasal washes were performed at 8 years. Associations between clinically significant respiratory disease (CSRD) and wheezing or rhinoconjunctivitis at 3, 5, and 8 years were estimated using multivariable logistic regression. Results Four hundred and twenty six children were followed of which 67.7% had at least one CSRD episode; 12% had respiratory syncytial virus (RSV)+CSRD and 36% had rhinovirus (RHV)+CSRD. All‐cause CSRD was associated with increased wheeze at 3 (OR 2.33 [95% confidence intervals (CI) 1.23–4.40]) and 5 (OR: 2.12 [95% CI 1.12–4.01]) years. RHV+CSRD was more strongly associated with wheeze at 3 years in STH‐infected (STH‐infected [OR 13.41, 95% CI 1.56–115.64] vs. uninfected [OR 1.68, 95% CI 0.73–3.84]) and SPT+ (SPT+ [OR 9.42, 95% CI 1.88–47.15] versus SPT‐ [OR 1.92, 95% CI 0.84–4.38]) children. No associations were observed between CSRD and rhinoconjunctivitis. Discussion CSRD was significantly associated with childhood wheeze with stronger associations observed for RHV+CSRD in SPT+ and STH‐infected children.


| BACKGROUND
Respiratory syncytial virus (RSV) is the leading viral cause of severe acute lower respiratory infections (LRI) in infants and children worldwide, causing up to 80% of bronchiolitis and an estimated >3.6 million hospitalizations and 84,500-125,200 deaths annually among children below 5 years of age. 1,2 Rhinovirus (RHV) is responsible for up to 20%-40% of medically attended acute wheezing and bronchiolitis in infancy, and is a leading cause of LRI outside the RSV season. Viral infections in infancy, particularly early LRI due to RSV and RHV, have been associated with recurrent wheezing and asthma in children. [3][4][5][6] Important questions remain about the directionality of the associations between viral LRI in infants and airway disease in children and potential interactions with atopy. [5][6][7][8] If causal associations do exist, the prevention of early viral LRI may reduce wheezing illness in early childhood and/or asthma in older children.
Although numerous studies over the last 30 years have evaluated these relationships, [9][10][11][12] few have been conducted in resourcelimited rural settings in low and middle-income countries where the burden of acute LRI is considered to be disproportionately high and a number of environmental factors might interact with early viral LRI to alter long-term wheezing and airway outcomes. 1 The hygiene hypothesis has postulated a protective role of common early childhood infections 13 and exposure to a wide diversity of microbes 14 against allergy and asthma. In recent years, there has been interest in the role of early life helminth infections in modulating the development of allergy or atopy and wheezing illness in childhood. [15][16][17][18] Soil-transmitted helminths (STH), primarily Ascaris lumbricoides and Trichuris trichiura, are estimated to infect over 1 billion humans worldwide. 19 A severe Th2-inflammatory response 20 associated with severe pulmonary disease 21 can be induced in mice experimentally infected with Ascaris, while potentially severe obstructive airway disease has been observed in humans with evidence of sporadic 22 or seasonal 23

exposures to Ascaris.
Early life exposure to STH parasites could modify the effects of early viral LRI on wheezing illness and asthma in later childhood through effects on airway inflammation. We followed a birth cohort in a tropical, rural region of Ecuador to investigate associations between clinically significant respiratory illness (CSRD) in the first 2 years of life and pulmonary outcomes at 3, 5, and 8 years in the context of endemic STH infections. We explored whether associations between CSRD and later respiratory outcomes might be modified by antenatal and early childhood exposure to STH parasites, and by early life endotoxin exposure and atopy at 2 years.

| Study population
This study was nested within the ECUAVIDA birth cohort, a study of 2404 children born between 2006 and 2009 in the district of Quinindé, Esmeraldas Province, in a rural tropical coastal region in northeastern Ecuador. ECUAVIDA was designed to evaluate the effects of a wide range of infectious and non-infectious factors on development of atopy and allergic disease to 8 years. 24 The cohort was recruited at the public hospital in the town of Quinindé, the only hospital serving the district. All newborns born at the hospital during the recruitment period were eligible for recruitment according to the following criteria: (i) healthy baby aged less than 14 days; (ii) resi-

| Routine procedures
Information on demographic, socioeconomic and household information, and respiratory symptoms were obtained using a questionnaire administered to the child's mother during routine visits around the time of birth of the child and at 3, 5, and 8 years. Relevant data were collected by questionnaire and physical examinations during clinic and home visits. 24

| Atopy
Allergen skin prick testing (SPT) was performed at 2 years for a panel of nine allergens (house dust mites, cockroach, dog, cat, fungi, grass pollens, egg, milk, and peanut) as described. 25 A positive test was a mean wheal diameter ≥2 mm greater than the negative saline control. SPT positivity was defined as reactivity to any allergen.

| Stool analyses
STH infections were detected by microscopy of stool samples collected from the mother during the third trimester of pregnancy and from children at 3, 7, 13, and 24 months using a combination of direct saline smears, Kato-Katz, formol-ether concentrations, and carbon coproculture. 24 26 Fractional exhaled nitric oxide was measured in parts per billion (ppb) using NObreath (Bedfont Scientific). Values greater than 35 ppb were considered elevated. 27 Nasal wash samples were collected and prepared as described 28 : slides with ≥5% eosinophils were considered eosinophilic.

| Nasopharyngeal swabs
Once respiratory surveillance was initiated, nasopharyngeal (NP) swabs were collected whenever children had symptomatic respiratory illness through a combination of active and passive surveillance through clinic and home visits for evaluation of acute respiratory illnesses. Routine telephone calls were made twice weekly to mothers to determine whether the child in the surveillance sample had respiratory symptoms, and medical records of visits to a dedicated cohort pediatric clinic were reviewed. Swabs were stored at −80°C and tested using multiplex rRT-polymerase chain reaction (PCR) (FTD Respiratory pathogens 21, Fast-Track Diagnostics, Luxembourg) for a range of respiratory pathogens including RHV and RSV.

| Endotoxin
Mattress samples for the measurement of endotoxin were obtained from the sleeping place of each child at 13 months using 1200W electric vacuum cleaners (Electrolux) and nylon filters (Indoor Biotechnologies, Inc.) to aspirate an uncovered area of 0.25 m 2 over 2 min. Dust samples were stored at −20°C prior to shipment for analysis of endotoxin levels (EU/mL) using the kinetic Limulus Amoebocyte Lysate assay (Indoor Biotechnologies).

| Definitions
Respiratory outcomes were defined: (1) CSRD-any respiratory illness with rales, wheezing, tachypnea, subcostal retraction, or nasal flaring detected on physical examination and/or a diagnosis of bronchiolitis, bronchitis, pneumonia; (2) recent wheeze-parental report of wheeze during the previous 12 months; (3) recurrent wheezing-parental report of wheezing on two or more occasions during the previous 12 months; (4) asthma-a parental report of wheeze over the previous 3 years at 8 years of age, and parentally reported wheeze up to 5 years or physician-diagnosed asthma at any time, or both; and (5) recent rhinoconjunctivitis-a parental report during the previous 12 months of relevant nasal symptoms in the absence of a cold and accompanied by itchy watery eyes. 29

| Bias
Stringent procedures to prevent losses to follow-up or incomplete data and sampling were implemented, including regular contacts by phone and home visits and maintaining a registry of neighborhood contacts. Reliance on maternal recall for the collection of questionnaire data could be subject to bias because of differential recall depending on maternal age and educational level. Data on a wide variety of relevant risk factors were collected to control for potential confounding. Exposures were measured using objective and standardized protocols (including laboratory methods for respiratory viruses, STH infections, and endotoxin) which will have minimized measurement bias. Data on outcomes were collected blind to exposure status. The original cohort study (of 2404 newborns) was designed to measure the effect of childhood STH infections on allergic outcomes and not for the effects of CSRD on wheeze and rhinoconjunctivitis. The sample size for this sub-sample of the cohort selected for respiratory infection surveillance was restricted by logistical and cost considerations. Our analysis of this sub-sample of the cohort with respiratory infection surveillance had limited power for detection of associations between infrequent exposures and or outcomes.

| Statistical analyses
The primary analysis examined associations between CSRD and wheeze at 3, 5, and 8 years. Secondary analyses addressed associations between CSRD and other respiratory outcomes (recurrent wheeze, asthma diagnosis, and markers of airways function and inflammation). Bivariate and multivariable logistic regression analyses were done to estimate associations with CSRD and potential confounders with outcomes. Potential confounders considered in the analyses are shown in Table 1. A socio-economic status index was created using principal component analysis as described. 17 Potential confounders in any of the bivariate analyses with p < 0.10 were kept in the final models using the same set of confounders to adjust all models. All multivariable models were controlled for sex. Potential a priori effect modifiers (maternal [any vs. none] and childhood [any vs. none during first 2 years of life] STH infections, and endotoxin exposure [>median vs. ≤ median of 18 EU/mL]) were evaluated using likelihood ratio tests. Significance was set at α = 0.05. Population fractions of CSRD attributable to individual pathogens were estimated as described. 30 Sensitivity analyses for uncertainty in estimates were explored including missing data assumed to be at random, and results did not qualitatively change the findings. All statistical analyses were performed using Stata 11 (Statacorp).   (15), any STH to 2 years (4), maternal allergic symptoms (4), maternal STH infections (5), and mattress endotoxin (34). Bold values denote statistical significance at the p < 0.05 level.

| Ethics considerations
ATWELL ET AL.

| Surveillance cohort participants
As shown in Figure 1, 504 children in the ECUAVIDA cohort were considered for participation in surveillance; 78 were excluded because of accessibility (i.e. lived outside a 10 km radius of Quininde).
Thus, 426 infants were included in surveillance. Follow-up was over 85% at each of the follow-up times from 3 to 8 years (Figure 1)

| Respiratory outcomes
Wheezing during the previous 12 months was observed in 18.0%, 18.6%, and 6.3% of children at 3, 5 and 8 years, respectively; for recurrent wheeze, the proportions at these time points were 10.6%, 11.3%, and 3.3%, respectively. Asthma was present in 9.1% of children

| Associations between CSRD and respiratory outcomes
Univariable associations between CSRD and individual, maternal, and household risk factors and recent wheeze at 3, 5 and 8 years of age are shown in Table 1 and multivariable associations are shown in

| Effect modification by endotoxin level, SPT, or STH infections
Multivariable associations between CSRD and recent wheeze at 3 years stratified by potential effect modifiers (i.e. maternal and childhood STH, endotoxin levels, and SPTþ at 2 years) are shown in Table 3. There was no statistical evidence of effect modification by any of these exposures on CSRD-recent wheeze associations. Analysis of associations between CSRD sub-groups and recent wheeze at 3 years stratified by the same variables are shown in

| DISCUSSION
Here, we used a surveillance sample nested within a birth cohort to explore the effects of CSRD associated with viral infections during the first 2 years of life on wheeze/asthma to 8 years and airway function and inflammation at 8 years. The cohort was recruited in a low-resource "pre-industrial" setting in a rural tropical district of coastal Ecuador where infections with soil-transmitted helminth parasites are endemic. 32 We explored the effects of exposures that could affect airway inflammation during the first 2 years of life, namely in utero and early childhood exposures to STH parasites, SPTþ, and endotoxin exposure in mattress dust. Our data provide evidence that CSRD, particularly when associated with RHV or mixed RHV/RSV infections, was associated with wheezing at 3 and 5 years.
There is some evidence that early childhood atopy and post-natal STH infections might enhance the effects of RHV on wheeze.
In this surveillance sample, the majority (67.7%) of children experienced CSRD during the first 2 years of life, substantial fractions of which were attributable to RSV (28%) or RHV (4.3%) infections. Wheezing illness was frequent before 8 years (≥18.0% at 3 and 5 years) but declined to below 6.3% by 8 years. CSRD was associated with recent and recurrent wheeze at 3 and 5 years, but the effects were not statistically significant at 8 years although In this study, the associations between CSRD and wheeze at 3 years were stronger among children with early STH infections compared with uninfected children, although the interaction was not T A B L E 3 Analysis of potential effect modification by key environmental exposures and individual characteristics on associations between clinically significant respiratory disease and wheeze at 3, 5, and 8 years of age.       year-round. High study retention (>85% until 8 years) should have minimized potential selection bias and detailed information collected on a wide variety of potential confounding factors should have limited uncontrolled confounding. We used CSRD to identify outpatient illnesses of the lower respiratory tract, given that hospitalization rates for lower respiratory illness would be expected to be low in a surveillance cohort of this size. CSRD likely represents a medically significant but non-severe illness of the lower respiratory tract.

Variable
In conclusion, our data from a population of children followed

This work was supported by PATH Vaccine Solutions and Wellcome
Trust (088862/Z/09/Z). We thank the ECUAVIDA study team, children, and their families for their participation in and support of the study.

CONFLICT OF INTEREST STATEMENT
The authors declare that they have no conflicts of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.